Gram negative bacteria, and some protozoa, are known to produce a cell surface glycolipid substance called lipopolysaccharide (LPS). Lipopolysaccharide is not a component of either human tissues or gram positive bacteria; thus, it is a marker specific to gram negative bacteria and a few pathogenic protozoa. In addition, at an estimated one million molecules per cell, it is the most abundant component on the cell surface of gram negative bacteria. All lipopolysaccharides possess two functionally distinct regions of structure: a fatty portion called "lipid A" and a carbohydrate section containing as few as 3 and up to as many as 350 and sometimes more sugar residues.
The carbohydrate portion of LPS may itself be divided into two structural entities: the core, generally composed of 8 to 11 sugar residues shared by all the members of a particular genus, and the O-side chain which is unique to a given species or a particular antigenic subgroup of a given speices. The majority of the antigenic sites on LPS are localized in the carbohydrates of the core and O-side chain. Antibodies specific for each portion of the LPS structure have been developed. An antibody specific for the core region of Salmonella LPS will react with Salmonella LPS regardless of the species from which it was derived, but will not react with LPS from other bacteria such as various Escherichia species. Antibodies specific for the O-side chain of a particular species of Salmonella will only react with members of that species and will not react with LPS from other species of Salmonella or other gram negative bacteria.
The lipid A portion of LPS possesses the majority of the physiological activity of LPS. Two such activities of lipid A are particularly important clinically. They are its ability to induce fever (pyrogenicity) and its ability to induce generalized vascular collapse (septic or endotoxic shock). Although improvements in treatment have resulted in better prognoses for many victims of shock, those suffering from endotoxic shock still have a mortality rate of 70 to 80% [Robins, Cotran, Pathologic Basis of Disease, W. B. Saunders Co., Philadelphia, PA, 140 (1979)]. Lipid A is a potent inducer of endotoxic shock. Clinical symptoms in humans typically result from levels as low as 3 to 4 pg of lipid A in LPS/mL of blood [Elin, et al., Bacterial Endotoxins: Structure, Biomedical Significance, and Detection with the Limulus Amebocyte Lysate Test, Ed. Cate et al., Alan R. Liss, Inc., New York, Pages 307-324 (1985)]. For a discussion of the Limulus amebocyte lysate test, see below. Thus, the structure of LPS can be summarized as having a carbohydrate region well suited to antigenic analysis and organism identification, and a highly conserved lipid A section possessing poor antigenic properties but potent physiological activity.
In addition to endotoxic shock, gram negative bacteria are frequently the etiologic agent of meningitis, urinary track infections, anaerobic abscesses, urethritis, food poisoning, and other human or veterinary ailments. For all of these conditions, there is a need to make an accurate and timely diagnosis so as to control the further spread of infection and to insure proper treatment. Generally, samples taken from a patient must be cultured for between 18 and 120 hours before a definitive diagnosis can be made. Often, physicians cannot wait for culture results before beginning patient treatment. This delay between the start of treatment and the receipt of culture results means that culture results serve to confirm or refute a presumptive diagnosis but do not influence the initial choice of patient therapy. The high cost of physician's malpractice insurance and the cost containment measures imposed by governmental diagnosis related groups (DRG's) has placed the practice of medicine under increased pressure to eliminate treatment with unncessary and potentially harmful agents. Thus, there is a need for a test that is sensitive, specific, and can provide information on which to make a diagnosis, within a time frame that will help in the initial choice of therapeutic agents. The primary need is to detect the presence of LPS produced by gram negative bacteria followed by genus/species identification.
One approach to meeting this need has been to develop immunoassays to detect antigens in clinical samples. Although ideally one would want to utilize an antibody to lipid A because of its highly conserved structure, no antibody with sufficiently high affinity and crossreactivity to be useful in an immunoassay has been found. The carbohydrate O-side chains contain many copies of the antigenic sites and can therefore bind many antibody molecules. This makes capturing and detecting these antigens in a sandwich immunoassay easy and very sensitive. However, O-side chains are restricted in their utility because of their extreme antigenic heterogeneity among microbial species. The carbohydrates of the inner core might be of greater utility due to their more conserved structure but generally the core structure is only capable of binding a single antibody making a sandwich immunoassay practically impossible.
One of the oldest tests for monitoring endotoxin (LPS) contamination is the rabbit pyrogenecity test. A sample of the material to be tested is serially diluted and aliquots of each dilution are injected into rabbits. The rabbit's temperature is then monitored. If the rabbit's temperature becomes elevated, the sample is deemed to be contaminated. An estimate of the quantity of LPS contamination is obtained as the reciprocal of the dilution at which no significant increase in temperature is observed in the rabbits. This assay is imprecise, requires large numbers of animals, and is of little use as a diagnostic procedure because it does not provide any information regarding the nature of the LPS or the organism from which it is derived. It is also expensive for industrial manufacturers to have to perform routinely, and has a limit of detection in the range of 100 ng LPS/mL liquid.
A rapid and sensitive test for the presence of LPS is the Limulus amebocyte lysate (LAL) test based on the work of Levin and Bang [Tai et al., J. Biol. Chem., Volume 252, 2178-2181 (1977)]. The horseshoe crab (Limulus polyphemus) possesses a primitive but effective defense against gram negative organisms. The LPS of the invading organism stimulates a series of reactions that culminates in the formation of a clot around the intruder. The enzymes and coagulogen proteins needed to form the clot are located in intracellular granules of the amebocytes that circulate in the blood of the horseshoe crab. When these amebocytes are separated from the blood by centrifugation, washed, and then lysed, the result is a clear and fluid cell lysate. In the LAL test, when this cell lysate is exposed to even picogram amounts of LPS, it becomes turbid and forms a gelatinous clot.
An improvement over the LAL test described above is the LAL chromogenic assay. This assay is based on the fact that gelation in the LAL test is the result of the action of a cascade of serine proteases. These proteases can be monitored directly in a non-LAL coagulation test format by adding a chromogenic substrate, such as those used in monitoring mammalian blood clotting enzymes, to the reaction mixture and observing color formation. Generally, this assay shows greater sensitivity than the LAL coagulation test and can be monitored using an analytical instrument rather than human observer.
Although both of the above LAL-based tests are extremely sensitive to LPS, and only LPS, and, therefore, to the presence of gram negative bacteria, they provide no information regarding the nature of the LPS or the organism from which the LPS is derived. In fact, the extraordinary sensitivity of Limulus amebocyte lysate to LPS limits its utility in the LAL test to those samples which contain the LPS of interest or to those samples in which the mere presence of any LPS is of interest. For many clinical specimens, for example a throat or rectal swab, commensal gram negative organisms would be present and, therefore, a positive Limulus lysate test would not necessarily indicate the presence of a potential pathogen. Furthermore, the Limulus lysate test cannot be performed on any sample that contains either a serine protease or an inhibitor of a serine protease, such as those involved in platelet aggregation and blood clotting, because such enzymes interfere with clot formation.
In an effort to determine whether a commercially available enzyme preparation was contaminated with endotoxin, Bryant et al. [J. Clin. Microbiol., Volume 17, 1050 (1983)] reported immobilizing Limulus amebocyte lysate on a microtiter plate. The lysate was immobilized by adsorption to the polystyrene wells using a standard carbonate-bicarbonate buffer, pH 9.6. The authors presumed that if enzyme was retained in LAL-coated wells, but not in blank, uncoated wells, the retention could only have been the result of the capture of complex formed between LPS contamination present in the sample and the enzyme. Since enzyme was, in fact, found to have been retained, the authors assumed LPS contamination.
LPS binding proteins are also known to be isolated from amebocytes present in the blood of Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpius rotundicauda. Lysates from these amebocytes can substitute for Limulus amebocyte lysates [Nakamura et al., Biochemica et Biophysica Acta, Volume 707, 217-225 (1982)].
The ubiquitous presence of gram negative bacteria in the environment also presents a challenge to manufacturers of biomedical supplies and cosmetics. The potency and clinical consequences of LPS contamination make it necessary that such products be certifiably free of LPS. This need has created a demand for fast, reliable, and inexpensive assays for the detection of endotoxins. There also exists a need to remove LPS from contaminated products.